This application claims priority of German Application No. 101 45 056.7, filed Sep. 13, 2001, the complete disclosure of which is hereby incorporated by reference.
a) Field of the Invention
The present invention is directed to an arrangement and a method by which the focus adjustment in microscopes with digital image generation, preferably in confocal microscopes, can be monitored and tracked.
b) Description of the Related Art
Various solutions are known from the prior art for determining and tracking the optimum focal plane in microscopes.
A method for determining the optimum focal plane evaluates the beam reflected by the object. The optimum focus position is achieved at maximum intensity of the reflected beam. For this purpose, according to WO 00/08415, a plurality of light spots with different focal planes are generated. The arrangement described in GB 2 321 517 for confocal microscopes also provides for the evaluation of the radiation reflected by the object for detecting the optimum focus position. These solutions are disadvantageous in that it is sometimes very time-consuming to determine the actual optimum focal plane. Numerous measurements are required for this purpose because the value of the optimum focus position can only be determined by approximation.
Further, solutions are known from the prior art in which light from a spectral range that is not used for examination is used for autofocusing. An example of this is the IR autofocus system. The essential disadvantage in solutions providing for the use of main optics for the IR autofocus system consists in that the main optics must be usable for a very broad spectral range which, in some cases, may extend from the DUV (deep ultraviolet) range to the IR range. An objective which is suitable for this purpose, such as that described in DE 199 31 949, can only be realized at a very high manufacturing cost.
A solution in which the autofocus system uses separate optics and not the main optics system is described in DE 199 19 804. This solution uses a secondary laser source for focus monitoring. The overall arrangement is substantially more extensive and complicated as a result of the additional arrangement for focus monitoring. Further, it is disadvantageous that the optimum focal plane must be known because the autofocus system only compares the instantaneous beam deflection to the ideal beam deflection.
Also, the technical expenditure in the solution described in WO 99/03011 is substantially increased by the additional optical elements required for focus monitoring. In this case, focus monitoring is carried out by the beam offset generated in defocusing by repeated reflection at the different optical surfaces.
The technical solutions known from the prior art have the disadvantages that the required additional light source results in a more complicated technical arrangement, that a large number of measurements are required and/or that focus monitoring is usually only possible with reflecting objects.
Therefore, it is the primary object of the present invention to develop a method and an arrangement suited to implement this method for focus monitoring in microscopes with digital image generation, preferably in confocal microscopes, without adding substantially to the complexity of the microscope construction and in which, if possible, the existing illuminating and image evaluating devices can be used to monitor the focal plane. The focus monitoring should be applicable independent from the (transparent, reflecting or fluorescing) object to be examined. Moreover, it should be possible to monitor the focal plane between normal work processes in a very fast and simple manner without altering the construction.
According to the invention, this object is met by the arrangement and the method for focus monitoring in that a suitably dimensioned rotatable plane plate is arranged in front of an existing main beam splitter in an area with parallel illumination beams, the axis of rotation of the plane plate being situated in relation to the specimen in such a way that a displacement of the beam bundle is carried out in the principal scanning direction or principal image direction during rotation. The images required for focus monitoring are recorded with a high zoom and with a pinhole having an aperture greater than one Airy. A first image is recorded with a plane plate inclined by angle +xcex1 and a second image is recorded with a plane plate inclined by angle xe2x88x92xcex1. These images are checked for correlation to the image contents in the direction of beam displacement by pixel-by-pixel displacement. The determined displacement xcex94s at optimum correlation is a measurement of the instantaneous focusing or defocusing. For a displacement xcex94sxe2x89xa00, defocusing is at a distance proportional to xcex94s.
The suggested method for focus monitoring and the arrangement provided for implementing this method are applicable with slight adaptations for all microscopes outfitted with digital image-generating methods and arrangements. In confocal microscopes, applicability to defocusing is limited in a depth range of about 5 to 8xc3x97xcex/NA2.
The invention will be described in the following with reference to two embodiment examples.